| Polysialic acid (PSA) is a homopolymer of sialic acid linked byα-2,8 orα-2,9 glycosidic (ketosidic) bonds. It is classified to highly linear polymer and has strong biodegradability and good biocompatibility. Polysialylation of protein therapeutics has led to markedly reduced proteolysis, retention of their activity in vivo, prolongation of their half-life in the circulation. Long PSA chain can be a suitable scaffold material in biomedical applications. PSA of different molecular weight can be used in defferent area. To control the quality and achieve an important prerequisite for the clinical application of PSA, it is necessary to explore the factors affecting the molecular of PSA in fermentation process and control its size.An end group assay method for determining the mean degree of polymerization of polysialic acid was developed. The total sialic acid was measured by resorcinol method and the end reducing group content of polysialic acid was determined by fluorospectrophotometry method. Then the mean degree of polymerization of polysialic acid is calculated by dividing total sialic acid content by the end reducing group content. The fluorescence reaction was conducted in boric acid buffer (pH 9.5) at 80°C for 25 minutes. The fluorescence intensity of the reaction was linear with the content of the end reducing group of polysialic acid. The range of the confidence interval was 1~20 mg/L sialic acids, giving a detection limit of 0.36 mg/L and a RSD of 3.7%, and the result was consistent with the HPGFC method.Effect of culture temperature and pH on the mean degree of polymerization of PSA during the batch culture of E.coli K235 was investigated in a 7 L fermentor. The results showed that when the pH value was maintained at 6.4, low temperature favour the biosynthesis of long chain PSA, but lower temperature will greatly affect the normal biosynthesis of PSA. When the culture temperature was controlled at 33oC, the mean degree of polymerization of PSA reached 252, which was 2.14 and 1.83 times, respectively, comparing with that of temperature controlled at 29oC and 37oC. When the culture temperature was controlled at 37oC, the PSA production reached 1.92 g/L, which was 2.59 and 1.27 times, respectively, comparing with that of temperature controlled at 29oC and 33oC. Also, when the culture temperature was controlled at 37 oC, the alkaline condition favoured the biosynthesis of long chain PSA, but slightly affected the amount of PSA concentration. When the pH of the medium was maintained at 7.4, the mean degree of polymerization reached 461, which was 11.0 and 1.83 times, respectively, comparing with that of pH maintained at 5.4 and 6.4. When the pH was maintained at 6.4, the production of PSA reached 1.92 g/L, achieveing the optimal PSA content comparing with the other conditions.Based on the above studies of the batch fermentation of polysialic acid, a two-stage fed-batch fermentation process with different pH control at different phases was developed. The pH condition was maintained at constant 6.4 at the logarithmic phase, and then was maintained at constant 7.4 in stationary phase. During the fermentation, ammonium sulfate and sorbitol was added at constant speed when ammonium sulfate was exhausted. The polysialic acid production reached 3.87g/L, the mean degree of polysialic acid reached 551 at the end of fermentation, and its molecular weight was about 170 kDa. |
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